绵羊肺炎支原体P130蛋白主要抗原域原核表达及其间接ELISA检测方法的建立  

Development of an indirect ELISA with the main antigentic region of Mycoplasma ovipneumoniae P130 protein expressed in E.coli

作　　者：王旭 张晓宇 张建华 黄海碧 王晓晖 史晓娜 郝永清 

机构地区：[1]内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室

出　　处：《中国兽医科学》2018年第3期281-287,共7页Chinese Veterinary Science

基　　金：内蒙古自治区科技计划项目“舍饲肉羊疫病动态防控关键技术研究”(201502070)

摘　　要：为建立绵羊肺炎支原体（Mycoplasma ovipneumoniae,Mo）快速检测方法,本试验以绵羊肺炎支原体膜蛋白P130为包被抗原建立间接ELISA抗体检测方法。根据基因功能预测注释,绵羊肺炎支原体（Mo）P130蛋白显示编码大小约为130.5 ku,本试验以Mo全基因组为模板,使用PCR扩增Mo P130的基因片段,根据生物信息学软件分析,选取P130主要抗原域P130-3（693 bp）,构建P130蛋白主要抗原域原核表达载体pET32a-P130-3,并转化至BL21菌,在0.8 mmol/L IPTG和37℃条件下诱导表达目的蛋白,经Ni-NTA亲和层析纯化后进行SDS-PAGE和Western-blot鉴定。以P130-3为诊断抗原,经过优化反应条件,建立检测Mo抗体的间接ELISA方法。结果表明,表达的P130-3蛋白经鉴定大小为43 ku,与预期值相符。P130-3-ELISA方法阴、阳判定临界值为：D（450）值=0.238,该方法与口蹄疫等阳性血清均无交叉反应,具有极强的特异性。组内变异与组间变异均小于5%,具有很好的重复性,利用该方法与兰州兽医研究所推出的间接血凝法对200份临床样本进行检测,两者的符合率在90%以上。本研究建立的Mo间接ELISA方法可用于Mo的诊断和流行病学调查。In order to establish a rapid detection method for Mycoplasma ovipneumoniae（Mo）,the indirect ELISA antibody detection method was established by using Mo membrane protein P130 as coating antigen.According to the prediction of gene function annotation,Mo encoding P130 protein display size is about 130.5 ku,this experiment using Mo genome as template,PCR amplification of Mo gene fragment using P130,according to the analysis of bioinformatics software,select the main antigenic domain of P130 P130-3（693 bp）,construction of the main antigenic domain of P130 protein in E.coli the expression vector pET32 a-P130-3,and transformed into BL21 bacteria,inducing the expression of target protein in 0.8 mmol L IPTG and 37 ℃,purified by Ni-NTA affinity chromatography followed by SDS-PAGE and Western-blot.Using P130-3 as the diagnostic antigen,the indirect ELISA method for detecting Mo antibody was established by optimizing the reaction conditions.The results showed that the expressed P130-3 protein was identified to be 43 ku in size,which was in accordance with the expected value.The critical value of P130-3-ELISA method was D（450）=0.238.The method had no cross reactivity with positive sera such as foot and mouth disease,and had strong specificity.The intra group variation and inter group variation were less than 5% ,with good reproducibility.Using this method and indirect blood coagulation method of Lanzhou Veterinary Institute,200 clinical samples were detected,and the coincidence rate of the two methods was above 90% .The Mo indirect ELISA method established in this study can be used for the diagnosis and epidemiological investigation of Mo.

关 键 词：肺炎支原体 原核表达 间接ELISA 

分 类 号：S852.62[农业科学—基础兽医学]